Training Course in Structural Biology:

NMR-Spectroscopy of biological macromolecules in liquid and solid state

Summary:
NMR-spectroscopy can yield information on structure and dynamics of biological macromolecules at atomic resolution, even in the absence of long-range order. NMR-spectroscopy in solution is already a well-established method for structure-determination, and recent developments in solid-state NMR spectroscopy have enabled structural studies also in systems which are neither crystalline nor soluble. In this course, we will give an overview about the principles of NMR spectroscopy and its applications in liquid-and solid-state NMR. Basic techniques in both disciplines will be presented.

Methods to be learned in the training course:
NMR spectroscopy of proteins in the liquid and solid phase

Supervisors:
Prof. Dr. Henrike Heise
Dr. Henrik Müller
Dr. Matthias Stoldt

Location:
Research Centre Jülich, D-52425 Jülich
Building 16.13, Ground level

During the time of the course, a shuttle bus will commute between the University and the Forschungszentrum Jülich.
A map of the bus stop can be found here.

BioStruct Fellows interested in this course must contact:
Prof. Dr. Henrike Heise: , +49(0)2461-61-4658, or
Dr. Henrik Müller: , +49(0)211-81-14703 , or
Dr. Matthias Stoldt: , +49(0)2461-61-5874

website:
http://www.biologie.uni-duesseldorf.de/Institute/Physikalische_Biologie/Gruppen/Arbeitsgruppe_Heise
http://www.fz-juelich.de/isb/isb-3/

Literature:
Keeler: Understanding NMR Spectroscopy, Cavanagh: Protein NMR Spectroscopy, T. Stangler, R. Hartmann, D. Willbold, and B. W. Koenig, Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules, Z. Phys. Chem. 220 (2006) 567–613, D.D. Laws, H.-M. L. Bitter, A. Jerschow, Solid-State NMR Spectroscopic Methods in Chemistry, Angew. Chem. Int. Ed. 2002, 41, 3096-3129. S.M. Luca, H.Heise, M. Baldus, High-resolution solid-state NMR applied to polypeptides and membrane proteins. Acc. Chem. Res. 2003, 36, 858-865.

Training Course in Structural Biology:

Fluorescence Spectroscopy and Microscopy: basics of fluorescence (CAI):

Summary:
Fluorescence is a widely used tool to investigate (bio-) molecules in in vitro and in vivo approaches. This course aims to communicate the basic principles of fluorescence. The phenomenon of fluorescence depends on several parameters. The interplay of these different parameters, that are relevant for the characterisation of a fluorescence probe, will be demonstrated in spectroscopic and microscopic experiments. Therefore we will investigate typical fluorescent molecules that are used in many different biomedical and biochemical applications like fluorescein, rhodamines, Alexa dyes, the green fluorescent protein, GFP, and fluorescently labelled antibodies. Furthermore we will also investigate the auto-fluorescence of proteins. The goal of our course is to enable the participants to use fluorescence as a reliable tool in their experiments and to accurately interpret their results.

Methods to be learned in the training course:

• Fluorescence spectroscopy
• Fluorescence correlation spectroscopy

Supervisors:
Dr. Ralf Kühnemuth
Dr. Stefanie Weidtkamp-Peters
Mr. Denis Dörr
Mr. Stefan Marawske

Location:
Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Building-No.: 26.32
Level: O2
Room: 25

BioStruct Fellows interested in this course must contact:
Dr. Stefanie Weidtkamp-Peters: , +49(0)211-81-14715
Dr. Ralf Kühnemuth: , +49(0)211-81-14864

Training Course in Structural Biology:

Fluorescence-based spectroscopic and calorimetric applications in the analysis of biomolecular interactions:

Summary:
To understand the mechanisms through which biological macromolecules interact with each other we need not only structural information, but also exact details of the kinetics and thermodynamics of the processes involved. This is particularly important for key proteins acting in signal transduction pathways such as the small GTPases of the Ras superfamily. The complexity of their roles is constantly increasing since a large number of GTPases have been identified and each of these in turn interacts with a variety of regulatory and effector proteins. There are a number of methods that can be used to characterize the specificity and strength of intermolecular interactions, to understand the effect of binding on defined protein structures, and, ultimately, to obtain insights into their biological functions. This training course will cover both theoretical and experimental approaches, including (i) fluorescence spectroscopy, which allows real-time monitoring of ligand– and protein–protein interactions at submicromolar concentrations, and quantification of the kinetic and equilibrium constants under cell-free condition as well as under cell-based condition using confocal laser scanning microscopy (cLMS), and (ii) isothermal titration calorimetry (ITC) that allows determination of thermodynamic parameters, including the free energy, enthalpy, entropy, and heat capacity of binding as well as the stoichiometry of the interaction and of course the binding constant.

Methods to be learned in the training course:

• Fluorescence labelling of ligands and proteins
• Fluorescence spectroscopy under cell-free and cell-based conditions
• Isothermal titration calorimetry

Supervisors:
PD Dr. Reza Ahmadian
Dr. Roland Piekorz
Ms. Mamta Jaiswal
Ms. Sarah Risse
Mr. Fabian Kuck
Mr. Badri Nath Dubey
Mr. Harish Chandra Thakur

Location:
Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Building-No.: 22.03
Level: O3
Room: 75

BioStruct Fellows interested in this course must contact:
PD Dr. Reza Ahmadian: , +49(0)211-81-12384

Training Course in Structural Biology:

Application of Electron Paramagnetic Resonance (EPR) Spectroscopy:

Summary:
In EPR spectroscopy, chemical entities that contain unpaired electrons can be observed and characterized. Such entities either are radical species or transition metals in various oxidation states. In proteins, both cases occur, which means that they either belong to the functional macromolecule or are transiently bound or appear in a reaction sequence (e.g. in electron transfer). Alternatively, radicals can be inserted or induced artificially into the protein of interest in order to make certain structural entities EPR accessible. Another possibility is the substitution of EPR silent metal ions by EPR active transition metals to probe the metal binding site of interest. At first hand, the data provided by EPR spectroscopy contain information about the electronic state of a paramagnetic centre. However, based on this, conclusions about the atomic structure can be drawn and distance determinations become possible. The accuracy of this structural information often exceeds that of other methods. In many cases, EPR spectroscopic data provide the only structural information, in particular when high-resolution crystals are not available and systems are too large for high resolution NMR spectroscopy. In many cases, EPR data complement the information gained by other structural methodologies. A number of different EPR techniques have been developed, reaching from continuous wave (cw) experiments to advanced pulsed EPR methods (for example ENDOR, PELDOR). This course will introduce a broad spectrum of current methods.

1. cw EPR and ENDOR spectroscopy of organic and biological radicals (9 GHz EPR at room temperature and liquid nitrogen temperature): Introduction into the EPR spectrometer; radicals in ash; organic radicals in solution; photochemically produced radicals (P+ in the reaction centre of bacteria, Fig. 1); nitroxide spin labels.
2. cw EPR of transition metal complexes in solution and solid state Introduction to anisotropic interactions. Single crystal study of a Cu complex.
3. EPR of photosystem II (PSII):
   Low temperature cw spectroscopy of synthetic and native (PSII) Mn complexes; further EPR signals of PSII.
4. PELDOR:
   Adjustment of pulse sequences, spectra simulation; Fourier transformation and filtering, interpretation of PELDOR spectrum.
5. 57Fe-Mößbauer spectroscopy:
   Introduction into the spectrometer; recording of the gamma spectrum und discrimination of the 14 keV radiation; sample preparation; recording of three different synthetic iron samples of unknown valence and spin states, at room temperature and at 80 K; interpretation of the spectra.

Supervisors:
Prof. Dr. Wolfgang Lubitz
Dr. Markus Knipp
Dr. Anton Savitzky
Dr. Eckhard Bill
Dr. Edward Reijerse

Location:
MPI for Bioinorganic Chemistry, Stiftstraße 34-36, D-45470 Mülheim a. d. R.

BioStruct Fellows interested in this course must contact:
Prof. Dr. Wolfgang Lubitz: , +49 (0)208 3063614, or
Dr. Markus Knipp:

Literature:

• Biophysik; W. Hoppe, W. Lohmann, H. Markl, H. Zigler, eds; Springer Verlag, Berlin, Heidelberg, NY 1977 (Kap. 3.3, S. 127-133)
• Biological Spectroscopy, I. D: Campbell, R. A. Dwek, Benjamin-Cummings Pub. Co; 1984 (Kap. 7)
• Biomolekular EPR-Spectroscopy; W. R. Hagen; CRC Press 2009

Training Course in Structural Biology:

X-ray crystallography: Why biochemistry needs X-rays and X-rays need biochemistry:

Summary:
Macromolecular crystallography of proteins is essential for the molecular understanding of proteins and their reaction mechanisms. This course will provide an overview about protein crystallography. In the practical course the advantages and difficulties will be shown and discussed. Ultimately the analysis of protein structures by their crystallographic data will allow the Biostruct-fellows to understand the role of protein crystallography in biochemistry.

Supervisors:
Prof. Dr. Lutz Schmitt
Prof. Dr. Georg Groth
Dr. Sander Smits
Dr. Daniel Schlieper

Location:
Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Building-No.: 26.42
Level: 03
Room-No.: 21

BioStruct Fellows interested in this course must contact:
Dr. Sander Smits: , +49(0)211-81-12647

Training Course in Structural Biology:

Fluorescence-based spectroscopic and calorimetric applications in the analysis of biomolecular interactions:

Summary:
To understand the mechanisms through which biological macromolecules interact with each other we need not only structural information, but also exact details of the kinetics and thermodynamics of the processes involved. This is particularly important for key proteins acting in signal transduction pathways such as the small GTPases of the Ras superfamily. The complexity of their roles is constantly increasing since a large number of GTPases have been identified and each of these in turn interacts with a variety of regulatory and effector proteins. There are a number of methods that can be used to characterize the specificity and strength of intermolecular interactions, to understand the effect of binding on defined protein structures, and, ultimately, to obtain insights into their biological functions. This training course will cover both theoretical and experimental approaches, including (i) fluorescence spectroscopy, which allows real-time monitoring of ligand– and protein–protein interactions at submicromolar concentrations, and quantification of the kinetic and equilibrium constants under cell-free condition as well as under cell-based condition using confocal laser scanning microscopy (cLMS), and (ii) isothermal titration calorimetry (ITC) that allows determination of thermodynamic parameters, including the free energy, enthalpy, entropy, and heat capacity of binding as well as the stoichiometry of the interaction and of course the binding constant.

Methods to be learned in the training course:

• Fluorescence labelling of ligands and proteins
• Fluorescence spectroscopy under cell-free and cell-based conditions
• Isothermal titration calorimetry

Supervisors:
PD Dr. Reza Ahmadian
Dr. Roland Piekorz
Ms. Mamta Jaiswal
Ms. Sarah Risse
Mr. Fabian Kuck

Location:
Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Building-No.: 22.03
Level: O3
Room: 75

BioStruct Fellows interested in this course must contact:
PD Dr. Reza Ahmadian: , +49(0)211-81-12384

Training Course in Structural Biology:

Structural bioinformatics course:

Summary:
The course aims at teaching widely used structural bioinformatics tools that allow predicting/modelling a protein structure, analyzing it with respect to structural, motional, and dynamic properties, and predicting a complex structure from its components. As a biological system, the well-characterized enzyme thrombin will be used. The course is designed as an integrated exercise, which allows re-using results from early stages in later stages. That way questions such as “How reliable are homology models for docking studies” can be addressed. In addition, comparisons to experimental data will be made frequently, such that students can learn the scope and limitations of the applied methodologies. The course consists of 3x2 hours of seminars and 4 days of practical course.

Methods to be learned in the training course:

• Homology modeling
• Molecular dynamics simulations
• Graph-theory based flexibility and rigidity analysis
• Molecular docking

Supervisors:
Prof. Dr. Holger Gohlke
Christoph Pfleger
Simone Fulle
Alexander Metz
Sina Kazemi

Location:
Seminars will be held in:
Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Lecture room 6B

Practical course will be held in:
Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Building-No.: 25.41
Level: O0
Room: 43

BioStruct Fellows interested in this course must contact:
Christopher Pfleger: , +49(0)211-81-12532

Additional information for BioStruct Fellows:
Date/periode:
Seminars, 3x: 19.5., 26.5., 2.6.2009, 8.15 – 10.00 am
Practical course: either 4.6. – 9.6.2009 or 12.6. – 18.6.2009, depending on availability.

The Course will be available also in the winter semester - exact dates will follow.

Training Course in Structural Biology:

X-ray crystallography: Why biochemistry needs X-rays and X-rays need biochemistry:

Summary:
Macromolecular crystallography of proteins is essential for the molecular understanding of proteins and their reaction mechanisms. This course will provide an overview about protein crystallography. In the practical course the advantages and difficulties will be shown and discussed. Ultimately the analysis of protein structures by their crystallographic data will allow the Biostruct-fellows to understand the role of protein crystallography in biochemistry.

Supervisors:
Prof. Dr. Lutz Schmitt
Prof. Dr. Georg Groth
Dr. Sander Smits
Dr. Daniel Schlieper

Location:
Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf
Building-No.: 26.42
Level: 03
Room-No.: 21

BioStruct Fellows interested in this course must contact:
Dr. Sander Smits: , +49(0)211-81-12647